Background: Neuroblastoma (NBL) is the most common extra-cranial solid tumour in childhood, with prognosis ranging from spontaneous remission to high risk for rapid and fatal progression. Despite existing therapy approaches, the 5-year event-free survival (EFS) for patients with advanced NBL remains below 30%, emphasizing urgent necessary for novel therapeutic strategies. Studies have shown that epigenetic disorders play an essential role in the pathogenesis of NBL. However, the function and mechanism of N7-methylguanosine (m⁷G) methyltransferase in NBL remains unknown. Methods: The expression levels of m⁷G tRNA methyltransferase Methyltransferase-like 1 (METTL1) were analyzed by querying the Gene Expression Omnibus (GEO) database and further confirmed by immunohistochemistry (IHC) assay. Kaplan-Meier, univariate and multivariate cox hazard analysis were performed to reveal the prognostic role of METTL1. Cell function assays were performed to evaluate how METTL1 works in proliferation, apoptosis and migration in cell lines and xenograft mouse models. The role of METTL1 on mRNA translation activity of NBL cells was measured using puromycin intake assay and polysome profiling assay. The m⁷G modified tRNAs were identified by tRNA reduction and cleavage sequencing (TRAC-seq). Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq) was utilized to identify the variation of gene translation efficiency (TE). Analyzed the codon frequency decoded by m⁷G tRNA to clarify the translation regulation and mechanism of m⁷G modification in NBL. Results: This study found that METTL1 were significantly up-regulated in advanced NBL, which acted as an independent risk factor and predicted poor prognosis. Further in NBL cell lines and BALB/c-nu female mice, we found METTL1 played a crucial role in promoting NBL progression. Furthermore, m⁷G profiling and translation analysis revealed downregulation of METTL1 would inhibit puromycin intake efficiency of NBL cells, indicating that METTL1 did count crucially in regulation of NBL cell translation. With all tRNAs with m⁷G modification identified in NBL cells, knockdown of METTL1 would significantly reduce the levels of both m⁷G modification and m⁷G tRNAs expressions. Result of RNC-seq shew there were 339 overlapped genes with impaired translation in NBL cells upon METTL1 knockdown. Further analysis revealed these genes contained higher frequency of codons decoded by m⁷G-modified tRNAs and were enriched in oncogenic pathways. Conclusion: This study revealed the critical role and mechanism of METTL1-mediated tRNA m⁷G modification in regulating NBL progression, providing new insights for developing therapeutic approaches for NBL patients.

MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R¹ position. Replacing the trifluoromethyl group on the phenyl ring at the R² position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.

MYCN and c-MYC are critical driver oncogenes in several childhood cancers, including neuroblastoma. Currently, the clinical development of MYC inhibitors has been hindered by the intrinsically disordered structure of MYC proteins, which lack well-defined ligand-binding pockets. Proliferation-associated protein 2G4 (PA2G4) directly binds to and stabilizes MYCN protein, leading to markedly increased MYCN levels in neuroblastoma cells. Here, we demonstrate that PA2G4 is essential for MYCN-driven tumor growth in neuroblastoma in vivo. Moreover, PA2G4 elevates c-MYC protein levels in neuroblastoma cells by inhibiting its ubiquitin-mediated degradation. In turn, c-MYC upregulates the transcription and protein expression of PA2G4, creating an oncogenic feed-forward expression loop. A small molecule PA2G4 inhibitor, WS6, directly disrupts the PA2G4-c-MYC protein–protein interaction, resulting in decreased levels of both PA2G4 and c-MYC. WS6 exhibited selective cytotoxicity in c-MYC-overexpressing cell lines. Together, these findings identify PA2G4 as a shared cofactor for both the c-MYC and MYCN oncoproteins and highlight its interaction with MYC family oncoproteins as a promising therapeutic vulnerability in MYC-driven cancers.

Background: Realgar-Indigo naturalis formula (RIF) containing A ₄ S ₄ as a major ingredient is an oral arsenic available in China. The efficacy of RIF on pediatric acute promyelocytic leukemia (APL) is comparable to arsenic trioxide (ATO). However, it remains to be explored that the effects of these two arsenicals on differentiation syndrome (DS) and coagulation disorder which are the two main life-threatening events in children with APL. Procedure: We analyzed 68 consecutive children with newly diagnosed APL involved in SCCLG-APL study (NCT02200978). Patients received all-trans retinoic acid (ATRA) on day 1 of induction therapy. ATO (0.16 mg/kg·d) or RIF (135 mg/kg·d) was administrated on day 5 after mitoxantrone on day 3 (non-high-risk group, NHR) or day 2-4 (high-risk group, HR). Results: The incidences of DS were 3.0% and 5.7% in ATO (n = 33) and RIF (n = 35) groups ( p = 0.590), and 10.3% and 0% in patients with and without differentiation-related hyperleukocytosis, respectively ( p = 0.04). The dynamic changes of WBC between the ATO and RIF groups were not statistically different. However, patients with high WBC counts or percentage of promyelocytes in peripheral blood tended to develop differentiation-related hyperleukocytosis. The improvement of coagulation indexes in the ATO and RIF groups had no statistical difference. Fibrinogen and prothrombin time had the quickest recovery rate. Conclusions: This study provides evidences that the incidence of DS and recovery of coagulopathy are similar whenever treating with RIF or ATO in children with APL.